Crispr epigenetic therapeutics for pain management

Abstract

Disclosed are effective, specific, and durable pain management therapies using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-based) epigenetic modulation of endogenous pathways involved in pain.

1 . A recombinant herpes simplex virus (HSV) vector comprising one or more polynucleotides encoding (a) at least one guide RNA comprising a guide sequence that hybridizes to a target sequence, and (b) a Cas nuclease. 
     
     
         2 . The HSV vector of  claim 1 , wherein the HSV vector is HSV-1. 
     
     
         3 . The HSV vector of  claim 1 , wherein the Cas nuclease is Cas9 nuclease. 
     
     
         4 . The HSV vector of  claim 3 , wherein the one or more polynucleotides encoding Cas9 protein comprise the nucleotide sequence of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41. 
     
     
         5 . The HSV vector of  claim 1 , wherein the target sequence is selected from the group consisting of SCN9A, MyD88, PENK, GAD1, KCNA2, and combinations thereof. 
     
     
         6 . The HSV vector of  claim 1 , wherein the guide sequence is selected from the group consisting of:
 (a) one or more of SEQ ID NOs: 1-7;   (b) one or more of SEQ ID NOs: 8-12;   (c) one or more of SEQ ID NOs: 13-20;   (d) one or more of SEQ ID NOs: 21-28;   (e) one or more of SEQ ID NOs: 29-35; and   (f) combinations thereof.   
     
     
         7 . The HSV vector of  claim 1 , wherein HSV vector comprises at least two guide RNAs. 
     
     
         8 . The HSV vector of  claim 1 , further comprising one or more polynucleotides encoding one or more repressors. 
     
     
         9 . The HSV vector of  claim 8 , wherein the one or more repressors are selected from the group consisting of Hp1a, Krab, MeCP2, and combinations thereof. 
     
     
         10 . The HSV vector of  claim 8 , wherein the one or more polynucleotides encoding one or more repressors comprise the nucleotide sequence of SEQ ID NO: 42 or SEQ ID NO: 43. 
     
     
         11 . The HSV vector of  claim 8 , further comprising one or more repression gRNA scaffolds. 
     
     
         12 . The HSV vector of  claim 1 , further comprising one or more polynucleotides encoding one or more activators. 
     
     
         13 . The HSV vector of  claim 12 , wherein the one or more activators are selected from the group consisting of VP64, p65, Rta, HSF1, and combinations thereof 
     
     
         14 . The HSV vector of  claim 12 , wherein the one or more polynucleotides encoding one or more activators comprises the nucleotide sequence of SEQ ID NO: 44. 
     
     
         15 . The HSV vector of  claim 12 , further comprising one or more activation gRNA scaffolds. 
     
     
         16 . The HSV vector of  claim 15 , wherein the one or more activation gRNA scaffolds comprise the nucleotide sequence of SEQ ID NO: 37. 
     
     
         17 . A pharmaceutical composition comprising the HSV vector of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         18 . A multiplexed CRISPR-based circuit comprising:
 (a) one or more guide RNAs (gRNAs) comprising (i) one or more guide sequences complementary to a portion of target sequence and (ii) one or more gRNA scaffolds;   (b) a nucleotide sequence encoding one or more repressors or one or more activators; and   (c) a nucleotide sequence encoding a Cas nuclease.   
     
     
         19 . A pharmaceutical composition comprising the multiplex CRISPR-based circuit of  claim 18  and a pharmaceutically acceptable carrier. 
     
     
         20 . A method for ameliorating pain in a subject, the method comprising administering to the subject a therapeutically effective amount of the HSV vector of  claim 1 , thereby ameliorating pain in the subject.